Viruses with linear, dsDNA genomes, such as the adenoviruses (Ad) and herpesviruses, encounter a number of host cell responses that may severely inhibit virus replication. This proposal focuses on the adenovirus E4-ORF3 protein which promotes virus replication by counteracting cellular antiviral responses. Adenoviruses have been recognized in recent years as significant pathogens in immunocompromised patients. There is no virus-specific therapy for Ad infection. It has become increasingly important, therefore, to fully understand host responses to Ad infection and viral strategies used to inhibit these responses in order to foster the development of antiviral therapies. The Ad E4-ORF3 protein functions by relocalizing a variety of cellular proteins into nuclear structures, referred to as tracks, to inhibit their activities. E4-ORF3 inhibits a DNA damage response and counteracts an interferon (IFN) response during infection. The E4-ORF3 protein induces the sumoylation of two cellular proteins, Nbs1 and Mre11, which are critically important effectors in a cellular DNA damage response. E4-ORF3 also binds SUMO and uses SUMO binding motifs to relocalize cellular proteins into nuclear tracks. These results place E4-ORF3 at the nexus of the cellular sumoylation system. Post-translational protein modification by SUMO regulates diverse cellular processes including transcription, DNA replication, DNA repair, subcellular localization, and ubiquitination. The induction of cellular protein sumoylation by E4-ORF3 likely is used to inhibit host antiviral activities. Studies are proposed to investigate the mechanism by which E4-ORF3 induces Nbs1 and Mre11 sumoylation, the functional consequences of these modifications, and identify and investigate other cellular proteins whose sumoylation is induced by E4-ORF3. The interaction of the E4-ORF3 protein with SUMO may represent the foundation by which E4-ORF3 recruits sumoylated cellular proteins into nuclear tracks and experiments are proposed to test this idea. The E4-ORF3 protein inhibits an IFN response during Ad infection by sequestering the proteins PML and Daxx, two prominent antiviral effectors. The mechanism(s) by which PML and Daxx inhibit Ad replication during the IFN-induced antiviral state will be investigated. The contribution of two other targets of E4-ORF3, transcriptional repressors TIF1 and TIF1, during an IFN response will be determined. The E4- ORF3 protein recruits a large number of different cellular proteins, associated with a variety of functions, into nuclear tracks. How this small viral protein accomplishes this is unknown. Physical analyses of wild type and mutant E4-ORF3 proteins will be conducted with the goal to study E4-ORF3 protein structure as it relates to function. Collectively, these studies will provide unique insight into cellular mechanisms that interfere with virus replication and viral responses that counteract these effects. PUBLIC HEALTH RELEVANCE: This research focuses on functions of human adenovirus which promote virus replication by counteracting antiviral host cell responses including an interferon response and a DNA damage response. Adenoviruses have been recognized in recent years as significant pathogens in immunocompromised patients. There is no virus-specific therapy for adenovirus infection, thus it has become increasingly important to fully understand host responses to adenovirus infection and viral strategies used to inhibit these responses in order to foster the development of antiviral therapies.